The Mycobacterium are a genus of bacteria that are characterized as acid-fast, non-motile, motile, gram-positive bacillus. The genus comprises many species including Mycobacterium afiicanum, M. avium, M. bovis, M. bovis-BCG, M. chelonae, M. fortuitum, M. gordonae, M. itracellulare, M. kansasii, M. leprae, M. microti, M. scrofulaceum, M. paratuberculosis, and M. tuberculosis. Some of the mycobacteria are pathogenic in both humans and animals, in particular M. tuberculosis, M. leprae, and M. bovis. Other mycobacterial species are not normally pathogenic, but cause opportunistic infections in immunocompromised individuals, such as AIDs patients. For example, infection by M. kansasii, M. avium, and M. intracellulare can cause severe lung disease in subjects in whom the immune system is suppressed or compromised. In fact, for the first time since 1953, reported cases of mycobacterial infections are increasing in the United States; many of these cases are related to the AIDS epidemic.
Conventional laboratory diagnosis of mycobacteria is based on acid-fast staining and cultivation of the organism, followed by biochemical assays. As a result of the slow growth and long generation time of mycobacteria, accurate laboratory diagnosis of mycobacteria by conventional techniques can take as long as six weeks. Automated culturing systems such as the BACTEC system (Becton Dickinson Microbiology Systems, Sparks, MD) can decrease the time for identification of mycobacteria to one to two weeks. Nevertheless, there still exists a need in the art to reduce the time required for accurate diagnosis of mycobacteria to less than a week, preferably to about one day.
Nucleic acid based diagnostic assays, such as Southern hybridization, offer rapid results, usually in less than one day. Polymerase chain reaction (PCR)-based methods for identifying mycobacteria are even more sensitive and can often provide results within hours. However, nucleic acid based methodologies for diagnosing mycobacteria are often fraught with drawbacks. Most of these methods are costly, are available for only a few species of Mycobacterium, and can resolve only one species per sample tested. Moreover, nucleic acid based assays require the development of oligonucleotide probes or primers that are specific for the genus Mycobacterium or for a particular species of Mycobacterium.
Isothermal amplification methods such as strand displacement amplification (SDA) and Self-Sustained Sequence Replication (3SR) have particular advantages in diagnostics, as they do not require the high/low temperature cycling characteristic of methods such as PCR. They are therefore simpler protocols and require less specialized equipment to perform. However, isothermal amplification methods such as SDA generally are not capable of amplifying targets as large as those amplifiable by PCR. Small target sequences severely restrict the ability to design primers and probes with the desired specificity for detection of a given target because the proximity of appropriate amplification primer binding sites becomes a factor, and there is less sequence available in the amplification product for assay probe design.
Initially, SDA was developed for use at temperatures between about 35.degree. C. and 45.degree. C. ("conventional SDA"). Recently, SDA has been adapted to higher temperatures using thermophilic polymerases and restriction endonucleases ("thermophilic SDA" or "tSDA") as described in EP 0 684 315 to Frasier et al. The tSDA system provides the advantages of increased speed and specificity as compared with conventional SDA. While the target binding sequences of amplification primers designed for use in conventional SDA generally will function in tSDA, they are usually shorter and amplification efficiency may therefore be reduced at the higher temperatures of tSDA. In contrast, primers comprising the target binding sequences of primers designed for tSDA usually function efficiently when adapted to amplification primers for conventional SDA or other amplification reactions.
To obviate the problems attendant to conventional diagnosis of Mycobacteinum, there have been attempts to develop nucleic acid based diagnostic methods using genus-specific hybridization or nucleic acid amplification with Mycobacteinum-specific oligonucleotides.
B. Boddinghaus et al. (J. Clin. Microbiol. 28, 1751 (1990)) disclose Mycobacterium genus-specific oligonucleotides derived from 16S rRNA sequences that specifically amplify and hybridize to mycobacterial DNA.
WO 95/31571 teaches oligonucleotides and methods for detecting species of Mycobacterium by ligase chain reaction. Oligonucleotides were selected from the DNA sequences of the M. tuberculosis protein antigen b gene, M. bovis IS987 direct repeat sequence, M. tuberculosis IS-like IS6110 element, M. tuberculosis 16S rRNA gene, M. tuberculosis 65 kDa heat shock gene, and the M. tuberculosis 10 kDa heat shock protein gene.
M. Hughes et al. (J. Clin. Microbiol. 31, 3216 (1993)) disclose methods of typing species within the genus Mycobacterium. Polymerase chain reaction with genus-specific primers is performed to amplify the 16S rRNA gene, followed by either restriction enzyme analysis or direct cycle sequencing to identify various mycobacterial species. These methods required 48 and 72 hours, respectively, to complete.
T. Rogall et al. (J. Gen. Microbiol. 136, 1915 (1990)) and P. Kirschner et al. (J. Clin. Microbiol. 31, 2882 (1993)) concern methods of identifying species of Mycobacterium using direct sequencing of PCR-amplified fragments of the 16S rRNA gene. Likewise, M. Vaneechoutte et al., (J. Clin. Microbiol. 31, 2061 (1993)) and E. Avaniss-Aghajani et al. (J. Clin. Microbiol. 34, 98-102 (1996)) teach methods of identifymg specific mycobacterial species by PCR amplification of a region of the 16S rRNA gene combined with restriction analysis of the amplification products.
U.S. Pat. No. 5,422,242 to Young teaches a two-step method for identifying species of Mycobacterium. First, genus-specific primers directed to conserved regions of the Mycobacterium rRNA gene are used to amplify a region of the 16S rRNA gene. Second, identification of particular species of Mycobacterium is performed by hybridizing species-specific probes directed to variable regions of the 16S rRNA gene to the PCR amplification products. Similar methods are disclosed by EP 0 528 306 and P. Kirschner et al. (J. Clln. Microbiol. 34, 304-12 (1996)).
Previous methods for detecting Mycobacterium in clinical specimens have suffered from unacceptably high variability in sensitivity, specificity, and incidence of false positives. See, e.g., G. Noordhoek et al., J. Clin. Microbiol. 32, 277-84 (1994); G. Pfyffer et al., J. Clin. Microbiol. 32, 918-23 (1994). Accordingly, there remains a need in the art for rapid, accurate and sensitive methods of identifying Mycobacteriwn.